Recent studies provide strong evidence that single class V myosin molecules transport vesicles and organelles processively along F-actin, taking several 36-nm steps, hand over hand, for each diffusional encounter. We demonstrated that the ATPase activity of myosin required calcium for maximal activity and showed that, in the absence of calcium, myosin V adopted a folded, inactive structure. We have used cryo-electron microscopy to examine the structure of myosin V that is walking on actin. These images give clear pictures of myosin V molecules with both heads attached to actin and will allow us to make observations about lever arm position and stiffness. We have used a fluorescently-labeled ATP analog termed deac-aminoATP to simulataneously visualize the stepping of fluorescently-labeled myosin V HMM and binding and dissociation of the nucleotide using the FIONA method. These studies show that there is a direct one to one coupling of myosin movement and nucleotide binding/dissociation and that there is strong gating between the kinetics of the two heads such that ADP release from the lead head is essentially prevented as long as the trail head is still attached.